The long-term goal of this study is to understand the molecular mechanisms by which a particular toxin-like protein binds to and penetrates into a cell. The problem concerns mechanisms of receptor function, protein translocation through one membrane, movement through an intermembrane compartment, binding to a second membrane, insertion into the membrane, channel formation, and voltage control of channel function. The toxin-like protein under discussion here is colicin E1. Solution to all of the above problems are of general relevance to the mechanism of action of toxins such as diphtheria and tetanus, and insertion of viruses into membranes. This similarity appears particularly graphic when the interaction of colicin E1 and diphtheria toxin with artificial planar membranes is compared. The two molecules show a very similar dependence for channel activity on pH, membrane potential, and lipid charge. Colicin El is a particularly good molecule in which to study all of these problems because it is naturally cloned, easily reclonable, and its sequence and that of its usual plasmid are known. Four aspects of the mechanism of action of colicin E1 will be analyzed: (1) Function of the colicin E1 receptor: Purification and characterization of the E1 receptor, binding of different colicin domains and peptide fragments to the receptor to localize the receptor binding site on the colicin polypeptide, incorporation of the receptor into liposomes, translocation of colicin through incorporated receptor. (2) Interaction of the C-terminal channel forming channel domain with membranes: pH dependence of binding, pH dependence of conformation in the presence and absence of membranes, role of a particular residue, glu 468, in the pH dependence, tested by chemical labeling and directed mutagenesis. The role of other residues in binding and control of voltage dependence would also be tested by directed mutagenesis. (3) Structure of colicin channel: Cleavage of exposed loops of channel forming polypeptide in liposomes, determination of Alpha-helical secondary structure content of colicin channel in liposomes, electron diffraction analysis of two dimensional membrane crystals of C-terminal fragment. (4) Mechanism of inhibition of channel formation by immunity protein: Use of artificial liposome system to study interaction of imm protein with colicin, question of whether imm protein acts from cytoplasm or inner membrane to block channel formation.